Svirskienė, Nataša
Item type:Publication, conference paper[2025][T1e][N010][1]; ; ; ; 17th International Conference of the Lithuanian Neuroscience Association „Brain Function, Dysfunction, and Translational Research“ : 28th November 2025, Kaunas, Lithuania, 2025-11-28, p. 61-61Glioblastoma (GBM) is an aggressive brain tumor marked by rapid growth, frequent recurrence, and resistance to treatment. Emerging research shows that GBM cells interact with neural stem cells (NSCs) through extracellular vesicles (EVs). Understanding how GBM-derived EVs influence NSCs is key to revealing tumor–microenvironment dynamics and identifying novel therapeutics. Because calcium signaling governs cell proliferation, migration, and differentiation, changes in intracellular calcium may provide an early indicator of EV-driven NSC reprogramming. This study examined how GBM cell lines from different origins affect NSC behavior, focusing on calcium-signaling changes and validating findings with next-generation sequencing (NGS). Human neural stem cells (NSC H9 line) were treated with EVs isolated from GBM cell lines A172 and U87-MG conditioned media using 12% polyethylene glycol (PEG) precipitation. Cells were exposed to GBM-derived EVs (~1250 EVs per cell) for 17 h for Ca2+ signalling analysis and for 24 h for NGS. For Ca²⁺ imaging, NSCs were loaded with 2 µM Oregon Green-488 BAPTA-1 dye and imaged at 2 Hz for 200 s using a 40× objective. Nifedipine (2 µM) and caffeine (5 mM) were applied sequentially to assess L-type Ca²⁺ channel activity and ryanodine receptor (RyR)-mediated Ca²⁺ release, respectively. The pilot study revealed that GBM EVs influence Ca²⁺ signaling in NSCs, with effects dependent on the cell line. A172 EVs increased Ca²⁺ signal frequency in affected NSCs, which was blocked by nifedipine, suggesting elevated L-type Ca²⁺ channel activity. In U87 EV– treated NSCs, signal amplitude increased with nifedipine and further rose with caffeine, indicating enhanced RyR activity. NGS analysis revealed changes in RyR, SLC8B1 and LETM1 gene expression, suggesting increased Ca²⁺ concentration in mitochondria leading to augmented metabolic activity in mitochondria. Changes in Ca²⁺ signaling induced by EVs point to potential cross-talk between cytosolic and mitochondrial Ca²⁺ pathways. The study shows that A172 EVs enhance Ca²⁺ signaling, potentially promoting NSC proliferation and differentiation, whereas U87-MG EVs strongly elevate RyR levels, increasing NSC sensitivity to environmental cues such as growth factors or inflammatory interleukins. These functional effects are further supported by NGS analysis, which confirms the underlying molecular changes driving these EV-mediated responses.
18 Item type:Publication, conference paper[2025][T2][N010][2]; ; ; Brain tumors 2025 : From biology to therapy : June 16-18, 2025, Warsaw, Poland : Abstract book, 2025-06-16, p. 64-65Background. Tumor-associated microenvironmental signaling plays a critical role in cancer proliferation by affecting adjacent healthy cells. Glioblastomas (GBMs), highly aggressive brain tumors, actively reshape their microenvironment through various mechanisms, notably via extracellular vesicles (EVs), which mediate intercellular communication. These EVs likely influence neural stem cells (NSCs), essential for brain homeostasis, neurogenesis, and repair. GBMs often arise near the subventricular zone (SVZ), rich in NSCs, suggesting potential two-way cellular interactions. Recent findings propose that NSCs may even serve as a cell of origin for GBM, though it remains unclear whether this is due to glioblastoma-induced signaling or intrinsic NSC traits. While SVZNSCs are implicated in GBM progression and recurrence, little is known about their direct response to tumor-derived signaling. This study aimed to explore how GBM cell lines of distinct origin affect NSC behavior, focusing on calcium signaling, a crucial second messenger involved in proliferation, differentiation, and cellular communication. Methods. GBM cell lines A172 and U87-MG were cultured in DMEM high-glucose media with 10% EV-depleted FBS for 72 h. EVs were isolated using 12% PEG precipitation and characterized via nanoparticle tracking analysis and ELISA for CD63, HSP70, and APOA1. NSC H9 cells were cultured in KnockOut DMEM/F-12 with StemPro supplement, bFGF, and EGF (20 ng/ml each), and stimulated with GBM EVs (~1250 EVs/cell) or control EVs from unconditioned media signaling was measured using Oregon Green-488 BAPTA- influx via L-type channels and RyR-mediated release from the endoplasmic reticulum. signaling change in NSC after stimulation with GBM EVs and its dependence on cell line EVs. The A172 EVs significantly increased the number signals in affected NSC by 25% compared to unaffected NSC (p<0.01 Kolmogorov-Smirnov, K-S, test), the difference was absent after application of nifedipine indicating increased levels of L-type Ca2+ channels. In NSC affected by U87 EVs Ca2+ signaling was not changed; however, the amplitude of signals was significantly increased by 29% after nifedipine (p<0.01, K-S test). The following caffeine application increased the amplitude by 16% (p<0.05, K-S test), and the number of signals increased by 126% (p<0.001, K-S test) indicating increased levels of RyR in NSC affected by U87 EVs. signaling is altered by GBM-derived EVs, affected by A172 EVs shows possible facilitation of NSC proliferation and differentiation. On the other hand, strong increase in RyR levels in NSCs due to U87-MG EVs shows potentiation of response of affected cells to possible influence by environmental molecular mediators of proliferation or differentiation, like growth factors, or inflammatory interleukins. Since transient Ca2+ signaling regulates cell differentiation, migration and proliferation, different effects of A172 and U87-MG EVs shows selective shaping of GBM cellular environment.
34 Item type:Publication, research article[2025][S1][N011,N010][9]; ; ; Archives of Biochemistry and Biophysics, 2025-06-07, vol. 771, p. 1-9Astrocyte function is controlled by intracellular Ca2+ signaling. On the other hand, hypoxia influences calcium dynamics and its homeostatic range because of reduction of ATP synthesis, which inhibits ATP dependent processes. By using Ca2+ sensitive fluorescence dye, we studied how metformin changed spontaneous oscillating Ca2+ signals in soma of astrocytes in monocultures, prepared from rat brains. Mild hypoxic conditions (2% O2) applied for 24 h had no effect on astrocyte viability; however, it reduced the relative amplitude of Ca2+ signals, slowed the decay of the signals, and increased the period of spontaneous oscillations. Lower concentrations of metformin, 250 μM or 500 μM, applied before hypoxia reduced this influence by partially restoring the amplitude, fastening the decay, and reducing the period of Ca2+ signaling. In contrast, higher concentration, 1mM of metformin exaggerated the effects of hypoxia by reducing signals, slowing their decay and prolonged the period between signals. Unexpectedly, in astrocytes grown under normoxic conditions, all concentrations of metformin after one hour of application had effects similar to hypoxia for Ca2+ signaling. In conclusion, our data show that mild hypoxia reduces Ca2+signaling in astrocyte cell monocultures, and low concentrations of metformin under mild hypoxic conditions help to rescue the functioning of astrocytes by conditioning the cells to prolonged hypoxic influence.
24WOS© Citations 1 Item type:Publication, conference poster[2024][T1e][N004][1]; ; ; 16th International Conference of the Lithuanian Neuroscience Association : 29th November 2024, Vilnius, Lithuania, 2024-11-29, p. 72-72Astrocyte function is controlled by intracellular Ca2+ signaling. On the other hand, hypoxia influences calcium dynamics and its homeostatic range because of reduction of ATP synthesis, which inhibits ATP dependent processes. By using Ca2+ sensitive fluorescence dye, we studied how metformin changed spontaneous oscillating Ca2+ signals in soma of astrocytes from 5–7-day-old rats grown under normoxic and mild-hypoxic (2% O2) conditions. Mild hypoxic conditions applied for 24 h did not change astrocyte viability; however, it reduced the relative amplitude of Ca2+ signals, slowed the decay of the signals, and increased the period of spontaneous oscillations. Lower concentrations of metformin, 0,25 or 0,5 mM, applied before hypoxia reduced this detrimental influence by partially restoring the amplitude, fastening the decay, and reducing the period of Ca2+ signaling. In contrast, higher concentration, 1mM, of metformin exaggerated the effects of hypoxia by reducing signals, slowing their decay and prolonged the period between signals. Unexpectedly, in astrocytes grown under normoxic conditions all concentrations of metformin after several hours of application had detrimental effects for Ca2+ signaling. Low concentration of metformin under mild hypoxic conditions helps to rescue the functioning of astrocytes by conditioning the cells to prolonged hypoxic influence.
10 Item type:Publication, conference paper[2024][T1e][N011,N004][1]; ; ; 4th Baltic Biophysics Conference (BBC) : Abstract Book : 2024 October 3-4th, Kaunas, Lithuania, 2024-10-03, p. 107-107Microglia are the main resident immune cells that are among the first responders to hypoxic/ischemic brain damages. Because of their sensitivity to blood flow fluctuations, microglia become activated and undergo morphological changes under hypoxic/ischemic conditions. However, intracellular mechanisms mediating microglial activation under hypoxic conditions are not well understood. When activated, microglia can cause a cascade of inflammatory processes and initiate cytokine release, activate reactive oxygen species production. It is known that intracellular Ca2+ signaling is linked with pathophysiological functions of microglia and its signaling changes, arising in response to brain damage. Previous studies found that calcium signaling is important for microglial immune function— cytokine release, P2X receptor trafficking and diffusion. Imaging of calcium-dependent fluorescence in vivo has demonstrated that microglial cells have spontaneous Ca2+ signaling which is important for microglial function. Spontaneous signals and their rate were changed by lipopolysaccharide and by local neuronal tissue injury. Also, changes in neuronal activity triggered increased microglial process Ca2+ signaling, which was related to process extension. However, there is still little knowledge on how Ca2+ spontaneous signals change in response to hypoxia. Metformin is widely used as anti-hyperglycemic agent for non-insulin-dependent (type 2) diabetes therapy. It has been suggested that metformin decreases hepatic glucose production mostly via inhibition of complex I of the mitochondrialrespiratory chain and activation of AMP-kinase signaling pathway. Recent studies suggest that metformin can also act as a neuroprotective agent during hypoxia/ischemia by suppressing mitochondrial complex I activity in neuronal cultures, by reducing ischemic stroke-induced oxidative stress, inhibiting neuronal apoptosis and suppressing neuroinflammation. Metformin has also been suggested to inhibit mitochondrial permeability transition pore (mPTP) opening and to exert neuroprotective effects by this mechanism. However, the link between mitochondrial dysfunction and inflammatory responses mediated by the activated microglia are not clear yet, and the role of metformin in these processes remains largely unknown. It has been demonstrated that metformin and phenformin at pharmacologically relevant concentrations differently improve Ca2+ homeostasis in hypoxia-affected primary cortical neuronal cell cultures, but little is known about their role in microglial cultures. In this study, we investigated the effect of metformin on spontaneous calcium signals in cultured microglia cells grown under normoxic and mild hypoxic conditions in order to elucidate the mechanism by which ischemichypoxic injury induce spontaneous calcium signaling changes in microglia. By using Ca2+ sensitive fluorescence dye, we studied how inhibition of mitochondrial respiration changed spontaneous Ca2+ signals in soma of microglial cells from 5–7-day-old rats grown under normoxic and mild-hypoxic conditions. In microglia under normoxic conditions, metformin or rotenone elevated the rate and the amplitude of Ca2+ signals 10–15 min after drug application. Addition of cyclosporin A, a blocker of mPTP, antioxidant trolox, or inositol 1,4,5-trisphosphate receptor (IP3R) blocker caffeine in the presence of rotenone reduced the elevated rate and the amplitude of the signals implying sensitivity to reactive oxygen species, and involvement of mitochondrial mPTP together with IP3R. Microglial cells exposed to mild hypoxic conditions for 24 h showed elevated rate and increased amplitude of Ca2+ signals. Application of metformin or rotenone but not phenformin before mild hypoxia reduced this elevated rate. Thus, metformin and rotenone had the opposing fast action in normoxia after 10–15 min and the slow action during 24 h mild-hypoxia implying activation of different signaling pathways. The slow action of metformin through inhibition of complex I could stabilize Ca2+ homeostasis after mild hypoxia and could be important for reduction of ischemia-induced microglial activation.
5 Item type:Publication, conference paper[2022][T1e][N004][2]; ; ; FEBS3+ Conference of Estonian, Latvian and Lithuanian Biochemical Societies : abstract book : Tallinn, Estonia, 15-17 June 2022 / Editor Tiit Lukk. Tallinn : Tallinn University of Technology Press, 2022. ISBN 9789949838639., 2022-06-15, p. 95-96.Hypoxic brain injury may affect neural tissue via microglia activation, however, mechanisms and consequences of these processes during hypoxia are not fully elucidated yet. We aimed to investigate effects of anti-hyperglycemic agent – metformin on developing brain microglia cells under normoxic or mild- hypoxic conditions. In this study primary rat microglial cultures (≥85 % microglia) at 7–11 DIV were treated with metformin (Met), cyclosporin (CsA) and rotenone (Ro). Cell cultures were incubated with or without pharmacological agents under normoxic and mild-hypoxic (93% N2, 5% CO2, 2% O2; 37°C) conditions for 24 h. It was shown that mild hypoxia (2% oxygen) had no effect on microglial cell viability which remained above 90%. None of Met concentrations (0,1mM; 0,5mM and 3mM) had effect on viability and number of microglial cells. CsA under hypoxic conditions tended to decrease both – cell number and viability, while Ro has no effect on number and viability of cells. Mild-hypoxia increases glutamate in microglia culture media, and pretreatment with Met but not Ro or CsA tend to reduce glutamate levels. We also found that none of the compounds effectively blocked mPTP opening in intact cells. Calcium dependent fluorescence measurements showed spontaneous calcium spikes; their generation was suppressed by CsA or trolox (0.1 mM), and enhanced by Ro, suggesting that Ca2+ spikes were mediated by mPTP opening. Hypoxia increased the frequency of Ca2+ spikes, while Met reduced the effect of hypoxia. These results suggest that hypoxia facilitates opening of mPTP in monotypic cell cultures and may cause release of glutamate into culture medium which may be reduced by Met.
6 Item type:Publication, journal article[2022][S1a][N010,T008][19]; ; ; ; ; Neuronal-glial cell cultures are usually grown attached to or encapsulated in an adhesive environment as evenly distributed networks lacking tissue-like cell density, organization and morphology. In such cultures, microglia have activated amoeboid morphology and do not display extended and intensively branched processes characteristic of the ramified tissue microglia. We have recently described self-assembling functional cerebellar organoids promoted by hydrogels containing collagen-like peptides (CLPs) conjugated to a polyethylene glycol (PEG) core. Spontaneous neuronal activity was accompanied by changes in the microglial morphology and behavior, suggesting the cells might play an essential role in forming the functional neuronal networks in response to the peptide signalling. The present study examines microglial cell morphology and function in cerebellar cell organoid cultures on CLP-PEG hydrogels and compares them to the cultures on crosslinked collagen hydrogels of similar elastomechanical properties. Material characterization suggested more expressed fibril orientation and denser packaging in crosslinked collagen than CLP-PEG. However, CLP-PEG promoted a significantly higher microglial motility (determined by time-lapse imaging) accompanied by highly diverse morphology including the ramified (brightfield and confocal microscopy), more active Ca2+ signalling (intracellular Ca2+ fluorescence recordings), and moderate inflammatory cytokine level (ELISA). On the contrary, on the collagen hydrogels, microglial cells were significantly less active and mostly round-shaped. In addition, the latter hydrogels did not support the neuron synaptic activity. Our findings indicate that the synthetic CLP-PEG hydrogels ensure more tissue-like microglial morphology, motility, and function than the crosslinked collagen substrates.
19WOS© Citations 8 Item type:Publication, journal article[2021][S1a][N004,N011][19]; ; ; International journal of molecular sciences. Basel : MDPI, 2021, vol. 22, no. 17., 2021-08-31, p. 1-19.Microglial functioning depends on Ca2+ signaling. By using Ca2+ sensitive fluorescence dye, we studied how inhibition of mitochondrial respiration changed spontaneous Ca2+ signals in soma of microglial cells from 5–7-day-old rats grown under normoxic and mild-hypoxic conditions. In microglia under normoxic conditions, metformin or rotenone elevated the rate and the amplitude of Ca2+ signals 10–15 min after drug application. Addition of cyclosporin A, a blocker of mitochondrial permeability transition pore (mPTP), antioxidant trolox, or inositol 1,4,5-trisphosphate receptor (IP3R) blocker caffeine in the presence of rotenone reduced the elevated rate and the amplitude of the signals implying sensitivity to reactive oxygen species (ROS), and involvement of mitochondrial mPTP together with IP3R. Microglial cells exposed to mild hypoxic conditions for 24 h showed elevated rate and increased amplitude of Ca2+ signals. Application of metformin or rotenone but not phenformin before mild hypoxia reduced this elevated rate. Thus, metformin and rotenone had the opposing fast action in normoxia after 10–15 min and the slow action during 24 h mild-hypoxia implying activation of different signaling pathways. The slow action of metformin through inhibition of complex I could stabilize Ca2+ homeostasis after mild hypoxia and could be important for reduction of ischemia-induced microglial activation.
11WOS© Citations 5 Item type:Publication, conference paper[2021][T2][N004,N010][1]; ; ; Virtual Federation of European Neuroscience Societies (FENS) Regional Meeting : Kraków, Poland, 25-27 August 2021 : book of abstracts / Federation of European Neuroscience Societies. Polish Neuroscience Society (PNS). Lithuanian Neuroscience Association (LNA). [Brussels] : Federation of European Neuroscience Societies, 2021., 2021-08-25, p. 157-157.Hypoxia may affect neural tissue via microglia, however, mechanisms and consequences of microglial activation during hypoxia are not well understood. We aimed to investigate effects of inhibitors of mitochondrial complex I – metformin and rotenone, and classical inhibitor of permeability transition pore (mPTP) on microglial cells. Primary rat microglial cultures at 7–11 DIV were treated with metformin (Met, 3 mM), cyclosporin (CsA, 10 μM) and rotenone (Ro, 5 nM) under normoxic or hypoxic (2% O2) conditions for 24 h. We show that 24 h hypoxia reduced number of microglia and had a cell viability- -reducing effect compared to normoxia. Met had no effect on viability and number of cells, CsA under hypoxic conditions tended to decrease both –viability and cell number, while Ro decreased cell viability but had no effect on microglia cell numbers. Hypoxia increased glutamate in microglia culture media, and pre-treatment with Met but not Ro or CsA tended to reduce glutamate levels after hypoxia. We also found that none of the compounds effectively blocked mPTP opening in intact cells. Calcium dependent fluorescence measurements showed spontaneous calcium spikes; their generation was suppressed by CsA or trolox (0.1 mM), and enhanced by Ro, suggesting that Ca2+ spikes were mediated by mPTP opening. Hypoxia increased the frequency of Ca2+ spikes, while Met reduced the effect of hypoxia. In conclusion, our results suggest that hypoxia facilitates opening of mPTP, slightly reduces microglial viability in monotypic cell cultures and causes release of glutamate into culture medium which may be reduced by Met.
15 Item type:Publication, journal article[2021][S1a][N004,N011][8]; ; ; Brain research. Amsterdam : Elsevier, 2021, vol. 1750., p. 1-8.Recent evidence suggests that metformin and phenformin may exert beneficial effects against neuronal injury in the ischemic brain, however, the difference of action between these two drugs and the molecular mechanism of such protection is not clear. In this study, we investigated whether mild hypoxia-affected neurons exhibit changes in cytosolic calcium handling and whether metformin and phenformin exert any effect on calcium homeostasis in hypoxia-affected neurons. Cultured primary rat cortical cells were stained with calcium sensitive dye Oregon Green 488 BAPTA-1,AM and spontaneous calcium dependent changes of fluorescence were recorded. Using obtained fluorescence traces we estimated changes in relative amplitude of recorded spontaneous signals, changes in frequency of spontaneous activity, and changes in decay of fluorescence traces. We found that hypoxia caused reduction of the relative signal amplitude, increased the spontaneous activity, and slowed the decay of calcium concentration. After pre-treatment of cells with 0.1-0.5 mM metformin, the relative signal amplitude increased and the frequency of spontaneous signals decreased in hypoxia-affected neurons. However, pre-treatment with 1-25 µM phenformin neither increased the relative signal amplitude nor reduced the frequency of spontaneous signals. The decay of fluorescence traces became faster after application of metformin or phenformin comparing to neurons under hypoxic conditions. These results suggest different action of metformin and phenformin in improvement of Ca2+ homeostasis in hypoxia-affected neurons, which may have different effects on neuronal survival and functions after hypoxia/ischemia.
8WOS© Citations 6